Abstract

Recent reports have indicated the participation of tight junction (TJ) proteins in the regulation of gene expression and cell proliferation. Here, we have studied the role of zona occludens (ZO)-2, a TJ peripheral protein, in the regulation of cyclin D1 transcription. We found that ZO-2 down-regulates cyclin D1 transcription in a dose-dependent manner. To understand how ZO-2 represses cyclin D1 promoter activity, we used deletion analyses and found that ZO-2 negatively regulates cyclin D1 transcription via an E box and that it diminishes cell proliferation. Because ZO-2 does not associate directly with DNA, electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) assay were used to identify the transcription factors mediating the ZO-2-repressive effect. c-Myc was found to bind the E box present in the cyclin D1 promoter, and the overexpression of c-Myc augmented the inhibition generated by ZO-2 transfection. The presence of ZO-2 and c-Myc in the same complex was further demonstrated by immunoprecipitation. ChIP and reporter gene assays using histone deacetylases (HDACs) inhibitors demonstrated that HDACs are necessary for ZO-2 repression and that HDAC1 is recruited to the E box. We conclude that ZO-2 down-regulates cyclin D1 transcription by interacting with the c-Myc/E box element and by recruiting HDAC1.

ZO-2 down-regulates cyclin D1 transcription. (A) Cyclin D1 promoter activity was tested under conditions of high ZO-2 levels. MDCK cells were transiently cotransfected with 1 μg of pXP2-CD1 reporter plasmid and the indicated amounts of pGW1-HA-ZO-2 (ZO-2) expression vector or with the empty vector (pGW1). pXP2-CD1/pGW1 is taken as control. (B) To test the specificity of the ZO-2 effect, MDCK sparse monolayers were cotransfected with different reporter plasmids (pRSV-β-Gal, pCAT-Sox, or pXP2-CD1) and ZO-2 or pCB6 ZO-1myc (ZO-1) as indicated in the graph. Control reference activity was taken for each reporter as the activity under the influence of pGW1. (C) Cells were transiently transfected with 10 nM siRNA designed to target ZO-2 mRNA or a NC siRNA not homologous to anything in the vertebrate transcriptosome. Forty-eight hours after initial transfection cells were retransfected with 1 μg of pXP2-CD1 reporter plasmid. Harvesting was performed after 72 h (total time). Graphs show relative luciferase activity taken as a percentage of control (only pXP2-CD1) activity. (D and E) In vivo transcription of cyclin D1 tested in response to differential expression of ZO-2. Total RNA was extracted from monolayers transiently transfected with full-length ZO-2 (pGW1-HA-ZO-2), the empty vector (pGW1; D), or transfected with siRNA for ZO-2 or NC (E). Semiquantitative RT-PCR assays were performed to detect cyclin D1 mRNA levels. Data were quantified by densitometry of amplified bands normalizing optical densities of cyclin D1 to those from the GAPDH signal. Data shown are relative to control cells transfected only with the empty vector. (F) Cyclin D1 protein levels in MDCK cells, transfected as indicated in D and E, were monitored by Western blotting. Total protein extracts from sparse MDCK cells, transiently transfected with full-length ZO-2 or the empty vector, were analyzed by immunodetection with specific antibodies against ZO-2, cyclin D1, and actin 48 h after transfection. Numbers to the left indicate the molecular sizes in kilodaltons. Data are the mean values ± SE of at least three independent experiments; *p < 0.05, Student's t test.

Overexpression of ZO-2 modifies cell proliferation rates. (A) DNA synthesis was monitored by [3H]thymidine incorporation into MDCK cells transfected with the ZO-2 expression vector (pGW1-HA-ZO-2) or the empty vector (pGW1). [3H]Thymidine uptake was determined at various posttransfection time points. Data shown are mean ± SE values of at least three independent experiments performed in triplicate. Differences are significant beginning at 24 h. (B) Wound healing assays were performed on confluent MDCK monolayers previously transfected with 5 μg of HA-pGW1-ZO-2 or the empty vector pGW1 or with 10 nM siRNA specific for ZO-2 or the NC. Wound closure was followed by taking microscopic images at different time points after monolayer damage was inflicted. In each case, denuded areas were evaluated, quantified, and compared with obtain the percentage of wound healing (see graph). Data obtained at wound time 0 were set as control. *p < 0.05, Student's t test.

ZO-2 repression is mediated by a putative E2F/E box located in the cyclin D1 promoter. (A) The effects of punctual mutations on cyclin D1 promoter, depicted as X in the TRE(AP-1) (change TGAGTCA→CGGTACC) and CRE (change TAACGTCA→TAAAGCTT) sites, were monitored by reporter assays done under a control condition and under the influence of ZO-2 overexpression. MDCK cells were transfected with 1 μg of pXP2-CD1, the TRE(AP-1) and CRE mutant constructs or one of these constructs together with 2 μg of pGW1-HA-ZO-2. Relative luciferase activities are expressed as a percentage of the activity of the control (pXP2-CD1 alone). (B) Cyclin D1 promoter constructs with diverse deletions over the E2F binding site were analyzed under a control condition and under the influence of ZO-2 overexpression. MDCK cells were transfected with 1 μg of pXP2-CD1 or the E2F binding site deletions constructs alone or together with 2 μg of pGW1-HA-ZO-2. Relative luciferase activities are expressed as a percentage of the activity of pXP2-CD1/pGW1 taken as the control. *p < 0.05, **p < 0.001, Student's t test. At the left side, mutagenesis sites and deletions are indicated. (C) In silico analysis of the cyclin D1 promoter sequence, located between −708 and −540, confirms the presence of an E box and reveals a putative E2F binding site. Chosen restriction enzymes are indicated.

ZO-2 associates in vivo to an E box element in the cyclin D1 promoter. (A) Schematic localization of specific primers used for PCR reactions in ChIP assays designed for the dog cyclin D1 promoter. Amplification of exon 1 was used as a negative control. (B) ChIP assays performed in sparse MDCK cells reveal the association of ZO-2 with an E box and not to the TRE(AP-1) element also present in the cyclin D1 promoter. The sizes of the amplified regions are 269 base pairs for the E box region, 200 base pairs for the TRE(AP-1) area, and 200 base pairs for exon 1. In this and the following ChIP assays, positive controls with H3Ac or H3 and negative controls with preimmune serum (PS) and no antibody (NoAb) were included. Input DNA, bound, unbound and no DNA fractions were amplified with the same set of primers as additional quality controls. (C) ChIP assay to detect recombinant ZO-2 were performed in cells that overexpress ZO-2 (pGW1-HA-ZO-2). Immunoprecipitates done with antibodies against ZO-2 and HA confirm the interaction of native and transfected ZO-2 (HA-tagged ZO-2) with the E box. Representative images of radioactive PCR products are shown. At least two independent experiments were performed for all ChIP assays.

c-Myc recognizes the E box region in cyclin D1 promoter. (A) Gel shift assays done with the indicated 32P-end–labeled oligonucleotide. Left, E box/E2F probe interaction with proteins present in the nuclear extract (NE) was competed (C stands for competitor added) with a nonlabeled E box/E2F oligonucleotide and E2F consensus or mutant oligonucleotides. Middle, E box/E2F labeled oligonucleotide was competed with consensus c-Myc probe, the corresponding mutant c-Myc or AP-1 as a heterologous competitor. Right, consensus c-Myc was used as labeled probe for reverse competition. Black arrowheads on the right indicate the position of the specific complexes (Cx). All competition experiments were performed in the absence or presence of 100-fold excess of the indicated nonlabeled competitor and 10 μg of nuclear extracts obtained from sparse MDCK cells. (B) Left, 60 μg of nuclear extracts obtained from sparse MDCK and HeLa cells was subjected to Western blot using an antibody against c-Myc. A 67-kDa band was detected. Right, ChIP assay reveals the in vivo association of c-Myc with the E box present in the cyclin D1 promoter. (C) c-Myc is present in ZO-2 immunoprecipitate. Input, 10% of immunoprecipitation starting material.

c-Myc cooperates with ZO-2 in down-regulating the cyclin D1 promoter. (A) Western blot done with c-Myc antibody reveals how c-Myc is overexpressed in MDCK cells transfected with the c-Myc-LTR/pBR322 or the empty vector LTR/pBR322 (indicated as pBR322). HeLa cell extract was included as a positive control. (B) c-Myc overexpression represses cyclin D1 promoter activity in a dose-dependent manner and displays specificity for the E box element. This assay was done with sparse MDCK cells transfected with 1 μg of the intact cyclin D1 promoter construct (pXP2-CD1) or with a plasmid lacking E box/E2F element (−492ΔCycD1) and indicated amounts of c-Myc-LTR/pBR322 plasmid or the empty vector LTR/pBR322. (C) Simultaneous overexpression of c-Myc and ZO-2 enhanced the repression of cyclin D1 promoter activity. MDCK cells were cotransfected as indicated with different combinations of the following plasmids: pXP2-CD1, ZO-2, pBR322, and c-Myc. (D) Addition of ZO-2 siRNA modifies c-Myc repression abilities. Over a constant c-Myc expression, ZO-2 siRNA but not NC interferes with repression. Recombinant ZO-2 expression abrogates the ZO-2 siRNA effect. Relative luciferase activities are expressed as a percentage of the control activity (pXP2-CD1/pBR322 or in combination with NC siRNA). Data are mean values ± SE of at least three independent experiments (*p < 0.05, **p < 0.001, Student's t test).

Myc/ZO-2 down-regulates cell proliferation. Cell proliferation assessed by [3H]thymidine incorporation (A) and by MTT method (B) in MDCK cells transiently cotransfected (electroporated) with 40 μg of combined vectors for 48 h as indicated in the figure. Results are expressed as a percentage of the empty vectors value (control). Bioassays were performed with each value being determined in triplicate. Mean values ± SE of at least three independent experiments are plotted. *p < 0.05, **p < 0.001, Student's t test. (C) Immunodetection of indicated proteins was performed as described in Figure 2 in the same cells electroporated as in A or B.

HDAC1 are recruited and participate in the negative effect mediated by ZO-2 and c-Myc on cyclin D1 transcription. (A) Participation of HDACs in the repression mediated by ZO-2/c-Myc. MDCK cells were transiently cotransfected with pXP2-CD1 reporter plasmid and pGW1-HA-ZO-2 and treated with HDAC inhibitors TSA (2 nM) and NaB (5 mM) for 12 h before harvesting. Results are presented as the percentage of repression exerted by ZO-2 under each treatment related to nontreated cells. Mean values ± SE of at least three independent experiments (*p < 0.05, Student's t test). (B) ChIP assay reveals the interaction of HDAC1 with the E box present in the cyclin D1 promoter. (C) HDAC1 is present in ZO-2 immunoprecipitate derived from nuclear extracts of sparse MDCK cells. A 60-kDa band was detected in the MDCK input and immunoprecipitation (IP) and in control HeLa cells. Input, 10% of immunoprecipitation starting material.